The post synaptic density (PSD) at excitatory glutamatergic synapses is a complex molecular machine which is known to be a key site of information processing and storage. In order to explore the detailed molecular organization of the PSD, we have developed a new method to freeze-substitute hippocampal cultures and then examine them by EM tomography in thin sections. Tomography reveals for the first time that the core of the PSD is a large array of vertically oriented filaments that contains PSD-95. We also identify two major type of transmembrane structures at PSD matching AMPA and NMDA receptors, which are both contacted by the PSD-95 containing vertical filaments. There are also two major types of filaments oriented parallel to the postsynaptic membrane. One type contacts the vertical filaments that also contact the NMDA receptors, while the other type contacts all vertical filaments. Some receptors in the spine membrane outside of the PSD are not contacted by filaments. The insight that the central organizing molecules, the lynchpin consisting of PSD-95 family proteins, are vertically oriented shows how its binding partners can form a stable matrix at the core of the PSD. The PSD-95 matrix can stabilize glutamate receptors and at the same time be remodeled by the addition of new receptors and their binding partners perhaps at the edges of the PSD. This hypothesis is now being tested by looking at the effects on the organization of the PSD by RNAi knocking down members of the PSD-95 family of proteins with EM tomography. When PSD-95 is knocked down much of the inner matrix of the PSD is missing, and in mice where PSD-95/93 is knocked out, fully mature synaptic spines are missing from key areas of the brain. We have developed an alternative preparation for tomography of isolated PSDs that is compatible with immunolabeling to identify components of the PSD, depending on the discovery of a negative stain compatible with tomography. A new project using mass spectroscometry is aimed at the quantification of PSD components from the highly purified preparation using a synthetic gene approach to produce recombinant artificial proteins with concatenated signature peptides. A covalent cross-linking strategy is applied to identify near neighbors at the PSD.